Non‐cytopathic bovine viral diarrhoea virus 2 induces autophagy to enhance its replication

Abstract Background Bovine viral diarrhoea virus (BVDV) is an important viral pathogen that has an economic impact on the livestock industry worldwide. Autophagy is one of the earliest cell‐autonomous defence mechanisms against microbial invasion, and many types of viruses can induce autophagy by infecting host cells. Objectives The aim of this study was to identify the role of autophagy in the pathogenesis of non‐cytopathic (ncp) BVDV2 infection. Methods Madin–Darby bovine kidney (MDBK) cells were treated with ncp BVDV2, rapamycin, or 3‐methyladenine (MA) and ncp BVDV2 and then incubated at 37°C for 24 h. Cells were harvested, and the effects of autophagy were determined by transmission electron microscopy (TEM), confocal laser microscopy, western blotting and qRT‐PCR. Apoptotic analysis was also performed using western blotting and flow cytometry. Results In ncp BVDV2‐infected MDBK cells, more autophagosomes were observed by TEM, and the number of microtubule‐associated protein 1 light chain 3B (LC3B) with green fluorescent protein puncta was also increased. The ncp BVDV2‐infected cells showed significantly enhanced conversion of LC3‐I to LC3‐II, as well as upregulation of autophagy‐related proteins, including ATG5 and Beclin 1, and substantial degradation of p62/SQSTM1. These results are similar to those induced by rapamycin, an autophagy inducer. E2 protein expression, which is associated with viral replication, increased over time in ncp BVDV2‐infected cells. Inhibition of autophagy by 3‐MA in ncp BVDV2‐infected MDBK cells downregulated the expressions of LC3‐II, ATG5 and Beclin 1 and prevented the degradation of p62/SQSTM1. Moreover, the expressions of phosphorylated Akt and procaspase‐3 were significantly increased in ncp BVDV2‐infected cells. In addition, the mRNA level of protein kinase R (PKR) was significantly reduced in ncp BVDV2‐infected cells. Conclusions Our results demonstrate that ncp BVDV2 infection induced autophagy in MDBK cells via anti‐apoptosis and PKR suppression. Therefore, autophagy may play a role in establishing persistent infection caused by ncp BVDV.


INTRODUCTION
Bovine viral diarrhoea virus (BVDV) causes significant economic losses worldwide in the cattle industry through decreased productive performance, immunosuppression and persistent infection. BVDV is a single-stranded positive RNA virus belonging to the genus Pestivirus, along with classical swine fever virus (CSFV) and border disease virus in the family Flaviviridae. Based on the 5′-untranslated region, two BVDV species have been identified: BVDV1 and BVDV2 (Baker, 1995). Each BVDV species is divided into two biotypes, cytopathic (cp) and noncytopathic (ncp), based on their ability to cause pathogenic effects in cultured cells (Ridpath et al., 2006). The ncp BVDV is the most prevalent biotype in nature and causes acute and persistent infections. In utero infection of cows during the first 120 days of pregnancy with ncp BVDV strains can result in the birth of persistently infected (PI) calves. These PI animals serve as a major source of viral spread in the herd (Brownlie et al., 1989;Darweesh et al., 2015;Polak & Zmudzinski, 2000). In contrast, cp BVDVs are relatively rare; however, when PI calves are superinfected with a cp BVDV strain, they may develop lethal mucosal disease.
These two viruses interact differently with the host innate immune response. The ncp BVDV evades the host adaptive and innate immune response to establish persistent infection. This may partly be due to the fact that ncp BVDV interferes with the induction of interferon (IFN) α/β (IFN type I) synthesis, the inhibition of which is associated with intracellular viral RNA accumulation (Baigent et al., 2002;Charleston et al., 2001;Gil et al., 2006;Schweizer & Peterhans, 2001;Vassilev & Donis, 2000). The lack of IFN production in ncp BVDV-infected cells might be advantageous for the survival of the virus, as it may prevent the stimulation of innate immune responses (Peterhans & Schweizer, 2014). In contrast, cp BVDV kills infected cells via apoptosis and induces IFN expression, which may be closely related to the execution of apoptosis (Grummer et al., 2002;Schweizer et al., 2006).
Autophagy is a quality-control system that degrades unwanted cytosolic components, such as damaged organelles and intracellular pathogens, and recycles the degradation products to maintain cellular homeostasis (Jordan & Randall, 2012;Zhou et al., 2017). Autophagy is also involved in viral pathogenesis and plays an important role in innate antiviral response. Interestingly, viruses have evolved to escape or use autophagic pathway for their own benefit; for example, many RNA viruses exploit autophagy for replication. Autophagosome formed during autophagy can provide a physical platform for viral replication machinery (Choi et al., 2018). Recently, complex interactions between autophagy and pathogens have been reported, and viral infections have been shown to induce autophagy, which serves as an innate immune mechanism against viruses, such as Sindbis virus and herpes simplex virus type 1 (Choi et al., 2018). However, the interplay between autophagy and viruses is extremely complex, and the success of many viruses depends on the subversion and sequestration of host autophagic responses (Deretic & Levine, 2009).
Recent studies have reported that ncp BVDV infection induces autophagy and significantly elevates the expression of autophagyrelated genes in Madin-Darby bovine kidney (MDBK) cells (Rajput et al., 2017;Zhou et al., 2017). However, the relationship between autophagy and ncp BVDV infection is not well understood. Therefore, this study aimed to investigate the mechanisms of autophagy induced by ncp BVDV2 and to identify the role of autophagy in the pathogenesis of ncp BVDV2 infection. These results provide useful information for understanding the pathogenesis of ncp BVDV2.

Cell culture and virus inoculation
MDBK cells and the virus strain were provided by the Animal and Plant Quarantine Agency (Gimcheon, South Korea) and cells were cultured in MEM-α supplemented with 10% heat-inactivated horse serum (Gibco, Waltham, MA, USA), antibiotic/antimycotic (Gibco) and 2 mM L-glutamine (Gibco) at 37 • C under 5% CO 2 . MDBK cells were tested to be negative for BVDV infection prior to being used in this study.
The virus strain used in this study was confirmed to be ncp BVDV2a by sequencing analysis (Seong et al., 2013), and this virus was low virulent. The ncp BVDV2a virus strain was obtained after 2 days of culture and centrifuged at 3000 × g for 10 min to remove cellular debris. The supernatant was frozen at −80 • C until used and virus titration was performed as previously described (Reed & Muench, 1938

Cell viability assay
MDBK cells (1 × 10 4 /well) were seeded in a 96-well plate and treated with ncp BVDV2, rapamycin or 3-MA and incubated at 37 • C for 24 h. Next, EZ-CYTOX (Daeillab, Changwon, South Korea) was added to each well according to the manufacturer's instructions and incubated at 37 • C for 2 h. After gentle shaking at room temperature for 1 min, the optical density was measured at 620 nm using a microplate reader (TECAN, Männedorf, Switzerland). Cell viability is presented as a percentage (%) relative to the control group (mock group).

Statistical analysis
Data are expressed as mean ± standard deviation. Each value represents the result of three independent experiments. Statistical analysis was performed using GraphPad Prism version 5.0 for Windows (Graph-Pad Software Inc., San Diego, CA, USA). Statistical significance was determined using two-or one-way analysis of variance post hoc followed by a least-squared difference test. p ≤ 0.05 was considered significant.

Ncp BVDV2 infection triggers autophagy in MDBK cells
To determine whether ncp BVDV2 can induce autophagy in MDBK

Ncp BVDV2 infection increases the levels of autophagy in MDBK cells
To investigate whether autophagy was induced by ncp BVDV2 infection, we first evaluated the expression of LC3 in ncp BVDV2-infected MDBK cells using western blotting. The conversion of LC3-I to LC3-II was monitored at 4, 12, 24, 36, and 48 h after ncp BVDV2 infection.

Ncp BVDV2 infection does not affect cell viability
The effects of autophagy regulators on cell viability were tested using the EZ-CYTOX assay. No significant differences in cell viability were observed after treatment with ncp BVDV2, rapamycin or 3-MA (p > 0.05), indicating that these treatments did not affect cell viability.

Autophagy induction reduces apoptosis
To further define the relationship between autophagy and apoptosis,
Many studies have shown that BVDV infection can induce autophagy (Fu et al., 2015;Rajput et al., 2017;Suda et al., 2019;Zhou et al., 2017). However, the specific role of autophagy in ncp BVDV2 infection has not been described. In the present study, our results demonstrate that ncp BVDV2 infection induces autophagy, resulting in enhanced virus replication, inhibition of apoptosis and production of IFN-mediated antiviral genes. Therefore, this study provides useful information that autophagy plays an important role in the pathogenesis of ncp BVDV2 infection.
Autophagy in mammalian cells is divided into six principal steps: initiation, nucleation, elongation, closure, maturation and degradation (Kang et al., 2011). In general, autophagosome, a double-membrane structure, is formed after the initiation of autophagy and engulfs cargo such as damaged organelles (Parzych & Klionsky, 2014). Subsequently, the autophagosome combines with the lysosome to form the autolysosome and digests its contents with lysosomal enzymes indispensable for autophagic vesicle formation, and its knockdown can result in reduction or total inhibition of autophagy, suggesting that it plays a central role in autophagy (Ye et al., 2018). During autophagy, cytosolic protein LC3-I is combined with phosphatidylethanolamine to become LC3-II, which is associated with autophagosomal membranes (Kabeya et al., 2000;Kuma et al., 2007). We found that ncp BVDV2 infection significantly enhanced the expressions of Beclin 1, ATG5 and LC3-II proteins. The increased expression of Beclin 1 and LC3-II is consistent with the results of previous studies . In the present study, the expression of ATG5 was observed in the ncp BVDV2-infected cells for the first time.

F I G U R E 3 Non-cytopathic (ncp) BVDV2 infection triggers autophagy in
ATG5, activated by ncp BVDV2, is believed to facilitate autophagosome membrane maturation. The levels of these autophagy-related proteins were reduced after 3-MA treatment of ncp BVDV2-infected cells; however, these changes were not significant. We further investigated another widely used autophagy marker, p62/SQSTM1, which binds directly to LC3 during autophagy and facilitates degradation of ubiquitinated protein aggregates (Jiang & Mizushima, 2015;Niklaus et al., 2017). Accumulation of p62/SQSTM1 indicates that autophagic flux is blocked (Klionsky et al., 2016). The results show the significant degradation of p62/SQSTM1 in ncp BVDV2-infected cells. This could be caused by the end of the degradation process of autophagy. Taken together, our findings provide clear evidence that ncp BVDV2 infection induces autophagy in the host cells.
Autophagy plays a pivotal role in the replication of several viruses.
The propagation of some viruses is suppressed by autophagy pathways, whereas other viruses exploit autophagy pathways to aid their replication (Ahmad et al., 2018). Additional studies are needed to determine the relationship between autophagy and viral propagation.
In this study, we found that ncp BVDV2 infection reduced apoptosis, Type I IFN is produced by cells in response to viral infection and induces an antiviral state through the regulation of protein synthesis and induction of ISGs (Sadler & Williams, 2008). Several studies have reported that cp BVDV induces IFN synthesis in host cells, whereas ncp BVDV does not, suggesting that this could be a defence mechanism to evade host innate immunity that might be critical for establishing persistent infection (Baigent et al., 2002;Charleston et al., 2001;Gil et al., 2006;Palomares et al., 2013). To date, the mechanism of inhibition of type I IFNs during ncp BVDV infection has not yet been completely resolved. Moreover, the role of type I IFNs in the persistent infection of BVDV remains unclear. During viral infection, a greater production of IFN may play an important role in virus clearance via the activation of innate immunity (Schmeisser et al., 2013). CSFV belongs to the Flaviviridae family and is also able to establish persistent infection. CSFV replication suppresses type I IFN-inducible antiviral activity and apoptosis by interfering with IFN production, thereby resulting in a persistent survival of the virus in host cells (Bensaude et al., 2004).
In the present study, among the IFN-mediated genes, such as Mx1, OAS1 and PKR, which have well-characterized antiviral activities, ncp BVDV2 infection significantly reduced the expression of PKR. This was associated with the inhibition of apoptosis. PKR has been reported to modulate a variety of cellular events, including apoptosis, antiviral state and cell growth rate (Gil et al., 2006). Suppression of PKR activation is critical for efficient replication of many viruses, delaying apoptosis and facilitating the establishment of persistent infection (Katze et al., 2002;Leib et al., 2000). 3-MA treatment in ncp BVDV2infected cells induced the increased expression of Mx1, OAS1 and PKR.
These effects were related to the reduction in autophagosome and induction of apoptosis. Consequently, our findings suggest that persistent infection caused by ncp BVDV may be closely associated with the suppression of PKR to evade the host immune response, resulting in enhanced viral replication via ncp BVDV2-induced autophagy.
Our results provide important evidence for establishing persistent infection.
In conclusion, the present study demonstrates that autophagy induced by ncp BVDV2 infection plays an essential role in viral replication and inhibits apoptosis and PKR activation. Consequently, autophagy may potentially play a role in establishing persistent infection caused by ncp BVDV. These findings expand our understanding of the pathogenesis of persistent BVDV infection and provide new insights into its control and prevention.